Understanding the control of human immunodeficiency virus (HIV) replication is of critical interest to the clinician hoping to deal with the AIDS epidemic, and the scientist seeking to understand the fundamentals of transcriptional regulation. The tat gene, which functions in trans to activate HIV gene expression, is one of the regulatory elements that plays a central role in the life cycle of this virus. As the effect of Tat on HIV expression is powerful and selective, greater knowledge about the structure and function of this activating protein could lead to effective and specific antiviral therapies. As Tat functions entirely in the context of eukaryotic transcriptional machinery, its study will shed light on basic mechanisms of transcriptional regulation. We propose to study four major biological questions with regard to Tat function. We will define the crucial cis-acting elements within the HIV long terminal repeat (LTR) that are required for Tat function by serially adding elements until a minimally function LTR is defined. The role of Sp1 will be particullarly examined by creating GAL4-Sp1 fusion proteins, so that critical domains of Sp1 can be defined. Study of activator fusion constructs, such as Tat-Rev and VP16-Tat, will dissect the molecular domains of Tat necessary for activation and interaction with DNA, RNA, and protein. Models of Tat function will be examined. Evidence that Tat is incorporated into transcriptional complexes will be sought by an immunoprecipitation/Southern blotting technique. In vivo footprinting will elucidate Tat interaction with DNA. A "bidirectional" promoter will be constructed to study the spatial constraints on Tat function. Finally, cellular factors that may modulate Tat function will be sought by affinity purification of proteins bound to a Tat-glutathione construct. It is hoped that thesae studies will yield information important to basic science and clinical medicine.